Experimental Study On The AngⅡ-induced MAPKs Signal Transduction And Its Intervention In Primary Cultured Rat Cardiomyocytes

Background and objective: Congestive heart failure (CHF) is a kind of serious syndrome threatening the health of patients with cardiovascular disease physically and mentally and is the end stage of various kinds of cardiovascular diseases, so it is still the main research area in basic medical and clinic field.It is well-known that CHF happens after the heart fails to function normally. Clinical studies have demonstrated that the cardiac hypertrophy is not only an adaptational state before cardiac failure, but is also an independent risk factor of cardiac vascular events. Thus it has become even more important to understand how cardiac hypertrophy develops. Cardiac hypertrophy is induced by increased mechanical load and many kinds of neural-humoral factors, such as Angll, endothelin-1, phenylephrine and peptide growth factors.Cardiac hypertrophy means the morphological change of cardiac tissue and re-organization of cardiac cells including the hypertrophy of myocyte and hyperplasia of fibroblast. Despite the understanding of cardiac hypertrophy is deeper than before, there are still many unclear aspects about myocytes hypertrophy, such as the assured and correlative pathway of signal transduction resulting in cardiac hypertrophy and the11mechanism controlling the signals and so on.It has already been definitely accepted that the locally produced Ang II is a more potent stimulator of the cardiac hypertrophy than circulating Angll. Moreover, it has been demonstrated that mechanical stress stimulates the secretion of Angll from cardiac myocytes and that Angll induces cardiac hypertrophy in cultured cardiac myocytes by autocrine mechanisms. Its effects are mediated through the angiotensin II type 1 receptor ( ATi-R) and the angiotensin II type 2 receptor (AIVR). Radioligand binding studies showed that type 1 and type 2 Angll receptors are present in near equal proportions in myocytes. Most of the Angll-stimulating effects is mediated by ATi-R and contributes to cardiac myocyte hypertrophy. The role of the AIVR subtype in the cardiac hypertrophy is even less well understood, although this receptor appears to serve as an antigrowth signal in proliferating cells. The two subtypes of Angll are all the members of G protein-coupled receptors.The intracellular signals are evoked with Ang II through seven transmembrane Ang II receptors which are G protein-coupled receptors. Among the signal molecules, ERK has recently attracted great attention of its importance in regulating cell growth. Stimulation of G protein-coupled receptors and receptor tyrosine kinases usually results in the activation of the Raf-MEK-ERK cascade in many cell types. In response to extracellular stimuli, ERK translocates from the cytoplasm to the nucleus, where ERK phosphorylates and activates several nuclear targets, ulteriorly ERK may play an important role in the pathogenesis of cardiac myocyte hypertrophy.Just based on the above, we used the primarily cultured12myocardiocytes, testing the myocyte viability, observing the state of phosphated ERK nuclear translocation and expression of c-fos mRNA and ERK under stimulating with Angll, aimed at understanding the influencing effects from different interventions and the mechanism of ERK in the nuclear translocation.Methods: Cardiomyocytes from neonatal rat were separated, cultured and identified primarily, and measured the myocyte viability with MTT method after irritating with Angll. We observed the action of Valsartan, CGP42112A and PD98059 to the Angll-induced viability changes. We used immunocytochemistry for observing the distributions of Phosphated ERK, RT-PCR for analysis of c-fos mRNA, and Western blot for semi-quantification of Phosphated ERK for activity of ERK.Results: 1. We made some modification to the traditional method in myocyte separation and culture by simple trypsin digestion. Through the record video, anti-actin immunocytofluorescence, it indicated we have harvested the higher than 90% purified cultured myocyte...